Particle Mixing

ABSTRACT

A method of separating an analyte from a biological sample is described. The method comprises providing particles capable of binding said analyte when present in a solution in a container, said container comprising walls, wherein at least a part of said walls is flexible. The particles are suspended in the solution by exerting a force on the flexible part of the walls of the container more than one time. An aliquot of the suspended particles is then removed from the container. The removed aliquot is dispensed into a sample, and the sample is incubated under conditions suitable to immobilize said analyte on the particles. The particles with the bound analyte are then separated from other material and at least part of the biological sample is removed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/946,417 filed on Nov. 19, 2015, which claims the benefit ofpriority to European Application No. EP 14193860.5, filed on Nov. 19,2014, the contents of which are incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The present invention relates to systems and methods to separate andanalyze biological molecules.

BACKGROUND OF THE INVENTION

Diagnostic assays for detecting or measuring analytes often require atleast partial purification of the analyte. One method commonly used inanalytical assays such as immunoassays or nucleic acid testing involvesbinding of the analyte either directly or indirectly to a particle.Thus, particles capable of either directly or indirectly binding theanalyte have to be provided to the sample to be tested. Particles may beprovided in a solid form, but preferably are provided as a suspension ofparticles in a solution, generally by transferring an aliquot of thesolution comprising the suspended particles using a pipetting device.

When such analyte-binding particles are provided as a suspension in asolution, care has to be taken that the suspension is homogenous when analiquot is aspirated by the pipetting device for transfer to the sample.Commonly, containers comprising a solution of such suspended particlesare subjected to shaking of the container using a shaking mechanism. Thehomogeneity of the suspended particle solution may be affected by theshaking movement itself carried out by the shaking mechanism and by thedesign of the container.

SUMMARY OF THE INVENTION

The present invention relates to a method of separating an analyte froma biological sample. The method comprises providing particles capable ofbinding said analyte when present in a solution in a container, saidcontainer comprising walls, wherein at least a part of said walls isflexible. The particles are suspended in the solution by exerting aforce on the flexible part of the walls of the container more than onetime. An aliquot of the suspended particles is then removed from thecontainer. The removed aliquot is dispensed into a sample, and thesample is incubated under conditions suitable to immobilize said analyteon the particles. The particles with the bound analyte are thenseparated from other material and at least part of the biological sampleis removed.

The present invention further relates to a system for separating ananalyte comprising a container. The container comprises walls. At leasta part of the walls is flexible. The container comprises particles in asolution. The system also comprises a separation station. The systemfurther comprises a movable mechanical device functionally coupled tothe flexible walls being part of the walls of the container, and acontrol unit configured to control the movable mechanical device suchthat it exerts pressure on the flexible walls being part of the walls ofthe container to suspend the particles in the container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a side view of the container of the present invention.

FIG. 2 shows a lateral view of the container of the present invention.

FIG. 3 shows that lid that covers the container and frame.

FIG. 4 is a schematic of the system of the present invention.

FIG. 5 is a schematic representation of a particle to which an analyteis bound.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to a method for mixing particles capableof binding a biological molecule. The method comprises the steps ofproviding particles suspended in a solution in a container, saidcontainer comprising walls, wherein at least a part of said walls isflexible, and suspending the particles by exerting a force on theflexible part of said walls of the container more than one time.

The term “analyte” as used herein may be any type of analyte which is ofinterest for detection, and the detection thereof is indicative of adiagnostic status of an organism. In one specific embodiment, theanalyte is a biological molecule. The organism can be animal or, in oneembodiment, human. “Biological molecules” may be proteins, polypeptides,antibodies or nucleic acids. In one embodiment, the analyte is a targetnucleic acid. A “target nucleic acid” is a polymeric compound ofnucleotides as known to the expert skilled in the art. “Target nucleicacid” is used herein to denote a nucleic acid in a sample which shouldbe analyzed, i.e. the presence, non-presence and/or amount thereof in asample should be determined. The target nucleic acid may be a genomicsequence, e.g. part of a specific gene, or RNA. In other embodiments,the target nucleic acid may be viral or microbial.

The analyte may be present in a liquid sample, or it may be present as asolid sample affixed to a support. Solid samples may include tissue.

The term “sample”, as used herein, refers to a material suspected ofcontaining an analyte of interest. The sample can be derived from anybiological source, such as a physiological fluid, including, blood,saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk,ascites fluid, mucous, synovial fluid, peritoneal fluid, amniotic fluid,tissue, cells or the like. The sample can be pretreated prior to use,such as preparing plasma from blood, diluting viscous fluids, lysis orthe like; methods of treatment can involve filtration, distillation,concentration, inactivation of interfering components, and the additionof reagents. A sample may be used directly as obtained from the sourceor following a pretreatment to modify the character of the sample, e.g.after being diluted with another solution or after having being mixedwith reagents e.g. to carry out one or more diagnostic assays like e.g.clinical chemistry assays, immunoassays, coagulation assays, nucleicacid testing, etc . . . The term “sample” as used herein is thereforenot only used for the original sample but also relates to a sample whichhas already been processed (pipetted, diluted, mixed with reagents,enriched, separated, having been purified, having been amplified etc).

In in-vitro diagnostics, the analyte is often enriched by separationfrom other material present in the sample. This enrichment is beneficialfor the sensitivity and the quality of a detection method. Oneestablished separation method comprises binding of a biological moleculeof interest to a solid support. The term “solid support” as used hereinrelates to any type of solid support to which the analyte is capable ofbinding, either directly and non-specifically by adsorption, orindirectly and specifically. Indirect binding may be binding of ananalyte to an antibody immobilized on the solid support, or binding of atag to a tag binding compound, e.g. binding of 6×His tags to Ni-chelate,or avidin binding to streptavidin coated beads. Solid support materialmay be a polymer, or a composition of polymers. Specifically, particlesmay be used as solid support material. Examples of particles includelatex particles, magnetic silica particles, metal particles, magneticglass particles. Other types of solid supports include glass fibers,glass fiber filters, filter paper, etc., while the solid supportmaterial is not limited to these materials.

When using particles as solid support for separation of an analyte, theymay be added in dry form to a sample. They may also be providedsuspended in a solution. When providing the particles suspended in asolution, the reproducibility of the method is affected by thehomogeneity of the suspension. It is, therefore, important to mix thesolution comprising the suspended particles just before transferring analiquot of this solution to the sample. An improved homogenous solutionis obtained by the present invention by providing the particlessuspended in a solution in a container which has walls of which at leasta part is flexible. The homogenous solution is obtained by exerting aforce on the flexible part of the walls of the container more than onetime. The force causes the liquid inside the container to move, therebyobtaining a mixing of the suspended particles, resulting in animprovement of the homogeneity of the suspended particles.

In one specific embodiment, the force is exerted onto a section locatedin the bottom half of said container, wherein said section is located inthe flexible part of said walls of said container.

In one embodiment of the invention, exerting of a force is achieved by amovable mechanical device. In one specific embodiment, said exerting ofa force is achieved automatically by a movable mechanical device. In oneembodiment, such a movable mechanical device may be a device with a toolconfigured to exert a force on a flexible part of the container. Thesurface contacting the container may be flat or rounded. The force maybe exerted e.g. by hitting or clapping the tool against a flexible partof the container. The movable mechanical device may be moved manually toexert a force on a flexible part of the container. For automation, thetool may be connected to a robotic arm. In either case, the force may beexerted by hitting the tool against a flexible part of the container.

In another embodiment, the force may be exerted manually. This may beachieved by squeezing the bottom part of the container with severalfingers, which then results in suspending the particles to obtain animproved homogeneity of the suspension of particles in the solution.

In one specific embodiment of the invention described herein, magneticparticles are used.

The present disclosure further relates to a container comprising walls,wherein at least a part of said walls is flexible. The containercomprises a solution comprising particles. The container is held in aframe, wherein said frame comprises an open bottom configured to permitaccess to the flexible part of the walls such that a force can beexerted on the flexible part of the walls of the container.

The term “frame” relates to an outer supporting structure for thecontainer. In one embodiment, the container is affixed to the frame. Theterm “affixed” relates to a physical attachment of the container to theframe. The container may be affixed by gluing or may be affixed byapplying heat. Other known types of affixing a container to a frame canbe used. The container is, thus, not integrally formed with the frame.Since the frame structurally supports the container, it is made of adifferent material than the container. In a specific embodiment, theframe is made of a stiff material.

The frame has an opening to permit access to a flexible part of thewalls such that a force can be exerted on the flexible part of the wallsof the container. In a specific embodiment, the frame has an open bottomto permit access to a flexible part of the walls such that a force canbe exerted on the flexible part of the walls of the container. Infurther specific embodiment, the frame has two long and two short walls,whereby one edge of each long side wall contacts one edge of a shortside wall. In a further specific embodiment, one of the long side wallsextends from the top end of the frame to a level above the bottom of theother long side wall. Thus, from the top downwards, one long side wallis shorter than the other side wall. This makes access for the movablemechanical tool easier. In one embodiment, the frame has an open topend.

In one embodiment, the container is closed on the bottom end andcomprises an open top end. The closed bottom end allows the container tohold the solution. The open top end allows the container to be filledwith solution and also makes it possible to remove the solution or analiquot thereof.

In one embodiment, the frame and the container comprise a common lid onthe top end. The lid, thus, covers the open top end of the container andthe open top end of the frame. In a specific embodiment, the lidcomprises at least one seal, wherein said seal is penetrable by apipette tip or a needle. The lid may be mounted on the frame andcontainer after the solution is filled into the container. The lidprotects the solution in the container from spilling or fromcontamination from the outside. In order to be able to remove an aliquotof the suspended particles from the lidded container and frame, at leastone seal is comprised on the lid which is configured to be penetrablefor a pipette tip or a needle.

A pipette tip is a tip, more specifically a disposable tip, which isreversibly or fixedly attached to a pipetting device. A needle is anon-disposable tip, specifically a steel needle, which may be reversiblyof fixedly attached to a pipetting device. These devices are used forpipetting. The term “pipetting” is herein used to indicate aspirating,i.e. withdrawing, a volume of liquid in a first step and dispensing avolume of liquid in a second step, wherein the volume of dispensedliquid may be the same or different from the volume of aspirated liquidand wherein intermediate aspirating and/or dispensing steps may or maynot occur between the first step and the second step. A pipetting deviceis an automated device which is configured to aspirate and dispenseliquids using pipette tips or needles. Such devices are well known inthe art. One pipetting device may be able to engage one or more tips orneedles, or may have one or more tips or needles fixedly attached to it.A penetrable seal is a seal which may be penetrated by a pipette tip orneedle. The seal may be frangible, i.e. the seal is broken by thepipette tip or needle and will, thereafter, not re-seal. In anotherembodiment, the seal is resealable. Examples of such resealable sealsare seals made of rubber. In one specific embodiment, the lid comprisesat least one split septum. A split septum is a seal which is pre-slit.Such seals are known in the art. They have the advantage that they canbe penetrated more easily than a septum which is not pre-slit. On theother hand, the split septum closes again after the pipette tip orneedle has been withdrawn, thus reducing the risk of spillage orcontamination of the contents of the container.

In one specific embodiment of the container herein described, thematerial of the container is a rubber-like material. Such a materialprovides flexibility to the walls, durability and makes it possible tovisually control the mixing and homogeneity of the particles suspendedin the solution contained in the container.

The present invention relates to a method of separating an analyte froma biological sample. The method comprises providing particles capable ofbinding said analyte when present in a solution in a container, saidcontainer comprising walls, wherein at least a part of said walls isflexible. The particles are suspended in the solution by exerting aforce on the flexible part of the walls of the container more than onetime. An aliquot of the suspended particles is then removed from thecontainer. The removed aliquot is dispensed into a sample, and thesample is incubated under conditions suitable to immobilize said analyteon the particles. The particles with the bound analyte are thenseparated from other material and at least part of the biological sampleis removed.

As mentioned above, in in-vitro diagnostics, an analyte is oftenenriched by separation from other material present in the sample. Thisenrichment is beneficial for the sensitivity and the quality of adetection method. One established separation method comprises binding ofa biological molecule of interest to a solid support. For this,particles capable of binding the analyte are provided in a container.Thus, the container holds a suspension of such particles. In order todispense a controlled amount of particles in an aliquot of thesuspension to a sample, the particles should be present as a homogenoussuspension in the solution contained in the container. The morehomogenous the suspension is, the more reliable and precise the data arewhich are eventually obtained from detecting or measuring the analytefollowing analyte separation. In the present invention, the homogenoussuspension is achieved by using a container comprising walls, wherein atleast a part of said walls is flexible, and by exerting a force on theflexible part of the walls of the container more than one time. By this,a homogenous suspension of particles can be obtained in the solution. Analiquot can then be removed from the solution in the container anddispensed into the sample. This ensures that an aliquot of a certainvolume will always contain the same amount of particles as the samevolume of a second aliquot taken from the same solution contained in thecontainer. Following dispensing of the aliquot, the sample is incubatedwith the particles under conditions suitable to immobilize said analyteon the particles. Such conditions are well known in the art. Once theanalyte is immobilized, the particles with the bound analyte are thenseparated from the other material present in the solution comprising thesample, and at least a part of the sample is removed.

The term “aliquot” as used herein relates to a portion of a solution.

In one embodiment of the method described herein, the methodadditionally comprises the step of washing said particles with the boundanalyte one or more times following separation from other material. Theterm “washing” is understood to mean that a volume suitable solution isadded to the particles with the bound particles. Suitable washingsolutions are wash buffers. A “wash buffer” is a fluid that is designedto remove undesired components, especially in a purification procedure.Such buffers are well known in the art. In the context of thepurification of nucleic acids, the wash buffer is suited to wash thesolid support material in order to separate the immobilized nucleic acidfrom any unwanted components. Thus, in one embodiment, the analyte is anucleic acid. The wash buffer may, for example, contain ethanol and/orchaotropic agents in a buffered solution or solutions with an acidic pHwithout ethanol and/or chaotropic agents as described above. Often thewashing solution or other solutions are provided as stock solutionswhich have to be diluted before use.

At the end of the washing step, at least a part of the washing solutionis separated and removed from the particles. In one specific embodiment,the washing step is repeated at least once.

In one embodiment, the method additionally comprises eluting the analytefrom said particles following separation or washing. The term “eluting”relates to the release of the bound analyte from the particles.Commonly, elution is performed using a suitable elution buffer. An“elution buffer” in the context of the invention is a suitable liquidfor separating analyte from the particles. Such a liquid may e.g. bedistilled water or aqueous salt solutions, such as e.g. Tris bufferslike Tris HCl, or HEPES, or other suitable buffers known to the skilledartisan. The pH value of such an elution buffer may be alkaline orneutral in the case that the analyte is a nucleic acid. Said elutionbuffer may contain further components such as e.g. chelators like EDTA,which stabilizes the isolated nucleic acids by inactivation of degradingenzymes.

The elution may require elevated temperatures. As a non-limitingexample, in case the analyte is a nucleic acid, the elution is carriedout at a temperature between 70° C. and 90° C., more specifically at atemperature of 80° C.

In one embodiment of the method, the force is exerted mechanically. In aspecific embodiment, the force is exerted by an operator by hand. Morespecifically, the operator may squeeze and release the flexible partrepeatedly by hand, thus exerting the mechanical force.

In another embodiment, the mechanical exerting is automated. In aspecific embodiment, the automation is achieved by a movable mechanicaldevice. In a more specific embodiment, the movable mechanical device maybe a device configured to hit or squeeze the flexible part of thecontainer. One specific embodiment of such a movable mechanical devicemay be a gripper or hammer head connected to a robotic arm.

In one embodiment of the invention, said removing of an aliquot from thecontainer is performed during exerting of force on the flexible part ofthe walls of the container. This has the advantage that the particlesare more homogenously suspended during the exertion of a force, andthat, thus, the aliquot contains a homogenous suspension of particles,thus increasing the reliability and quality of the detection ormeasurement of the analyte, which is ultimately done.

The present invention further relates to a system for separating ananalyte comprising a container. The container comprises walls. At leasta part of the walls is flexible. The container comprises particles in asolution. The system also comprises a separation station. The systemfurther comprises a movable mechanical device functionally coupled tothe flexible walls being part of the walls of the container, and acontrol unit configured to control the movable mechanical device suchthat it exerts pressure on the flexible walls being part of the walls ofthe container to suspend the particles in the container.

Systems for separating an analyte using particles capable to bind ananalyte are well known in the art. Such systems comprise a separationstation. When using particles which are not magnetic, a suitableseparation station may comprise a filter unit which is configured toretain the particles and a vacuum system which is configured to removethe liquid from the particles when a vacuum is applied. Anotherembodiment of such a separation station is a centrifugation unit whichis configured to pellet the particles within a vessel containing thesample and the particles with bound analyte, thus, separating theparticles from the solution present in the container. Another type ofseparation station which is commonly used is a magnetic separationstation configured to retain magnetic particles with bound analyte atthe bottom or on the walls of one or more vessels containing a sampleand particles with bound analyte. All of these separation stations areconfigured to separate particles with bound analyte from the sample andthe liquid component of the particle suspension, and to remove thesample and other liquid after binding the analyte to the particles. Theparticles can then be either washed and recovered, or directlyrecovered. As mentioned before, the particles thus separated from theother material in the sample may then additionally be washed to removecontaminants, and/or the analyte may finally be eluted from theparticles. The thus obtained analyte may then be detected or measured inthe same or in a different system.

The system of the present invention comprises a movable mechanicaldevice functionally coupled to the flexible walls being part of thewalls of the container. The term “functionally coupled” is understood tomean that the movable mechanical device interacts with the flexible partof the walls in a manner that permits obtaining a homogenous suspensionof the particles in the solution contained in the container.

The system comprises a control unit (CU) for controlling the analyzer.Such a control unit may be a separate unit or may be an integral part ofan analytical instrument. The control unit controls the analyzer in away that the necessary steps for the assay protocols are conducted bythe analyzer. That means the control unit e.g. instructs the analyzer toconduct certain pipetting steps to mix the sample with reagents or thecontrol unit controls the analyzer to incubate the sample mixtures for acertain time and so on. The control unit receives information from thedata manager which test has to be done with a certain sample and basedthereon determines the steps the analyzer (and maybe also a samplepreparation unit) has to perform. In certain embodiments the controlunit might be integral with the data management unit or may be embodiedby a common hardware.

In one embodiment of the system of the present invention, the containeris held in a frame, wherein said frame comprises an opening at thebottom of the container. In a specific embodiment, the frame and thecontainer are covered by a lid. In a more specific embodiment, the lidcomprises at least one opening. This opening can permit access to thecontents of the container In an even more specific embodiment, aresealable seal covers said opening. Such seals are made of a flexiblematerial, e.g. rubber or a thermoplastic material. They preventcontamination or spillage of the contents.

In one embodiment of the system, it further comprises a pipettingdevice. In a more specific embodiment, the pipetting device and saidmovable mechanical device are functionally coupled and said control unitis configured to control the movable mechanical device and the pipettingdevice such that the movable mechanical device exerts a force on theflexible walls being part of the walls of the container to suspend theparticles in the container for at least the same time period that thepipetting system requires to aspirate an aliquot of the solutioncomprising the particles, and said movable mechanical device exerts aforce on the flexible walls being part of the walls while the pipettingsystem aspires an aliquot of the solution comprising particles.

The term “the pipetting device and said movable mechanical device arefunctionally coupled” means that their pipetting function and exerting aforce functions are coordinated.

EXAMPLES

FIG. 1 shows a container (1) comprising a closed bottom end (7) and anopen top end (9). The open top end (9) of the container (1) is coveredby a lid (2). The container (1) comprises walls (6). At least a part ofwalls (6) are flexible walls (3). The container (1) contains a solution(4) in which particles (8) are suspended. A force can be exerted on theflexible part (3) of the walls (6) of the container (1) by a movablemechanical device (5) to achieve homogenous mixing of the particles (8).

FIG. 2 shows the container (1) comprising walls (6), at least part ofwhich comprise flexible walls (3). The container (1) further comprises aclosed bottom end (7) and an open top end (9). Like in FIG. 1, thecontainer (1) contains a solution (4) in which particles (8) aresuspended (not shown in this Figure). The container (1) is held in aframe (10). Frame (10) comprises an opening (11) on the bottom (12). Themovable mechanical device (5) can access the flexible walls (3) ofcontainer (1) through the opening (11) of the frame (10). In onespecific embodiment, the movable device (5) may comprise a robotic arm(13).

FIG. 3 shows a lid (14) which covers container (1) (not shown) and frame(10). In one specific embodiment, the lid (14) comprises openings (15)in which a resealable seal (16) can be set. FIG. 3 shows, as a specificembodiment, a septum strip (17) comprising a base (18) and multiplesepta (16). Each resealable septum (16) is penetrable for a pipette tip(19) which is attached to a pipetting device (20). The pipette tip (19)may be a disposable tip releasably attached to the pipetting device (20)or a needle affxed to the pipetting device (20).

FIG. 4 is a schematic of a system (23) comprising a separation station(22), a pipetting device (20) and a movable mechanical device (5). Thesystem (23) further comprises a container (1) with a solution (4)comprising particles (8). A control unit (21) comprised in the system(23) is configured to control the movable mechanical device (5) and thepipetting device (20) such that the movable mechanical device (5) exertsa force on the flexible part (3) of the walls (6) of the container (1)to suspend the particles (8) (not shown in this Figure) in the container(1) for at least the same time period that the pipetting device (20)requires to aspirate an aliquot of the solution (4) comprising theparticles (8) (not shown in this Figure).

FIG. 5 is a schematic representation of a particle (8) to which ananalyte (24) is bound.

1. A system for separating an analyte comprising a container, saidcontainer comprising walls, wherein at least a part of said walls isflexible, wherein said container comprises particles in a solution, anda separation station, the system further comprising a movable mechanicaldevice functionally coupled to said flexible walls being part of thewalls of said container, and a computer controller configured to controlthe movable mechanical device such that it exerts pressure on theflexible walls being part of the walls of the container to suspend theparticles in the container, wherein the system further comprises apipetting device, characterized in that said pipetting device and saidmovable mechanical device are functionally coupled and said computercontroller is configured to control the movable mechanical device andthe pipetting device such that the movable mechanical device exerts aforce on the flexible walls being part of the walls of the container tosuspend the particles in the container for at least the same time periodthat the pipetting system requires to aspirate an aliquot of thesolution comprising the particles, and said movable mechanical deviceexerts a force on the flexible walls being part of the walls while thepipetting system aspires an aliquot of the solution comprisingparticles.
 2. The system of claim 1, wherein said container is held in aframe, wherein said frame comprises an opening at the bottom of thecontainer.
 3. The system of claim 2, wherein said frame and saidcontainer are covered by a lid.
 4. The system of claim 3, wherein saidlid comprises at least one opening.
 5. The system of claim 4, wherein aresealable seal covers said opening.